This invention relates to Ubiquitin peptide extensions which serve as substrates for assaying for enzymes that modify peptide chains. More particularly, this invention relates to cloned Ub-peptide extensions as novel substrates for assaying protein kinases or enzymes that modify the C-terminus of the Ha-RAS protein.
Among the enzymes left to be discovered and characterized, many catalyze reactions in which proteins are the substrate. These classes of enzymes include the ever-expanding families of protein kinases (see Edelman et al., Ann. Rev. Biochem (1987) 56:567-613 and Hunter, Curr. Opinion Cell Biol. (1989) 1:1168-1181). The central importance of protein kinases in controlling cell behavior becomes so evident with each passing year that this point now needs little emphasis (see Hunter et al., Annu. Rev. Biochem. (1985), 54:897-930; Edelman et al., supra; Taylor, Bioessays(1987), 7:24-29; and Kikkawa et al., Annu. Rev. Biochem., (1989), 58:31-44 for review). What remains surprising, however, is the large number of different kinases present in eucaryotic cells.
Kuenzol et al., J. Biol. Chem., (1987), 262:9136-9140; Meggio et al., FEBS Lett., (1988), 237:225-228; Abdel-Ghany et al., Proc. Nat. Acad. Sci. USA, (1988), 85:1408-1411; Yasuda et al., Biochem. Biophys. Res. Commun., (1990), 166:1220-1227 and Litchfield et al., FEBS Lett., (1990), 261:117-120 all have shown that synthetic peptides can be used both to assay protein kinases and to deduce the amino acid sequences required for phosphorylation by the enzymes. Deana et al., Biochim. Biophys. Acta, (1990), 1051:199-202 demonstrates that synthetic peptides also permit assay of protein phosphatases.
Other enzymes that catalyze reactions in which proteins are the substrate include enzymes that add lipids to proteins (see Glomset, et al., TIBS (1990), 15:139-142 and Gordon, et al., J. of Biol. Chem. (1991), 266:8647-8650) and a wide variety of enzymes that modify specific amino acid side chains on cellular and secreted proteins (see Clark, Ann. Rev. Biochem. (1985), 54:479-506, Hart et al., Ann. Rev. Biochem. (1989),58:841-874 and Rechsteiner, Ubiquitin (1989) Plenum Press, 346 pp.
Examining the substrate specificity of that many enzymes is a formidable task. Analysis of such enzymes requires peptides, either as potential substrates for measuring kinetic features of these catalysts or in the preparation of affinity matrices for purifying them. Means for identifying and assaying enzymes per se is a commercial venture in which many business entities are engaged. Enzymes are used in detergents, in diagnostic testing, as keys to biotech operations. It is likely that enzymes will be used on an industrial scale in future applications.
In this regard, small peptides obtained as Ub extensions offer distinct advantages over smaller peptide substrates. First, they are less expensive to synthesize in large quantities. Also, Ub-peptides can, in certain cases, be purified from bacterial extracts by simple acid extraction. Some enzyme assays may require electrophoretic analysis for accurate quantitation and the fact that Ub-peptides migrate on SDS-PAGE gels in a region (.about.9K) virtually devoid of other proteins that may also become modified is a distinct advantage of the use of Ub-peptide extensions over the peptide alone. Furthermore, the presence of Ub at the N-terminus of the peptide substrate is thought to be crucial for product capture by PDVF or other electroblotting membranes. Another important advantage of ubiquitin-peptide extensions is the availability of proteases able to remove precisely the peptide extension from ubiquitin without further cleaving the released peptide.
The present invention therefore depends to a large extent on ubiquitin, a remarkable small protein with a variety of useful properties. Ubiquitin is a highly conserved, 76-residue protein having a C-terminus composed of arg-gly-gly and is found in all eucaryotic cells both free and covalently conjugated to a variety of cellular proteins. Ubiquitin is found in cells as diverse as mammals, yeast and celery. Ubiquitin is attached by its carboxyl terminus to amino groups of other proteins. When ubiquitin is attached to the alpha-amino terminus, such products are referred to in the literature as ubiquitin carboxyl extension proteins. In eucaroytic cells, the extension proteins are cleaved from the ubiquitin molecule by hydrolases (peptidases). It has been postulated that attachment of ubiquitin to a protein is a signal for the latter's destruction by proteolysis. Ubiquitin is lacking in cysteine and tryptophan but there is nothing unusual about its sequence other than its extreme conservation. The identical sequences of animal ubiquitins differ only at three positions from the yeast and plant ubiquitin. The x-ray structure of ubiquitin, which has been resolved to 2.8 A, reveals a compact globular protein with the carboxyl terminal arg-gly-gly extended into the solvent. The molecule contains four strands of (beta)-sheet plus a single (alpha)-helix with three and one half turns; all sequence differences among species being located on a small portion of the ubiquitin surface. NMR studies have shown that ubiquitin remains folded at pHs of 1 to 13 and below 80.degree. C. A distinct hydrophobic core and extensive hydrogen bonding are present, which may account for the molecule's exceptional stability. Ubiquitin has a neutral isoelectric point of 6.7 and a molecular weight of 8565. Ubiquitin is not only stable to extremes of pH, as noted above, but also to heat.
For a detailed analysis of ubiquitin, its properties and functions, reference is made to the book "Ubiquitin", published by Plenum Press, N.Y. (1989) and edited by Martin Rechsteiner.
Ubiquitin is an extremely soluble protein that can be expressed to very high levels within E. coli cells as shown by Butt et al., Proc. Nat. Acad. Sci. USA, (1989), 86:2540-2544. Carboxyl terminal peptide extensions are easily and cheaply prepared by cloning in E. coli. For example, the preparation of synthetic ubiquitin peptide fusion products containing up to about forty additional amino acid residues as ubiquitin extensions expressed in procaryotic cells, such as in E. coli, is described by Rechsteiner et al., copending patent application Serial No. 07/420,544 filed Oct. 12, 1989 and by Yoo et al., J. Biol. Chem. (1989) 264:17078-17083. Another important advantage of ubiquitin-peptide extensions is the availability of ubiquitin hydrolase enzymes able to remove precisely the peptide extension without further cleaving the released peptide.
Because some of the Ub-peptide extensions described herein contain a modified ubiquitin, a 76-residue protein, in which the last three amino acids of the C-terminus, composed of arg-gly-gly in native ubiquitin, have been changed to prevent cleavage by the ubiquitin hydrolase enzymes, the term Ub.sub.73 is sometimes used to designate the N-terminal 73 residues of ubiquitin. When the term "Ub-" followed by a specific designation is used, the ubiquitin referred to can be either the native 76 residue molecule or a molecule in which the last three residues of arg-gly-gly have been modified, as in the Ub-AEX designation. However, it will be clear from the specific designation used whether the ubiquitin is native or modified at the last three residues.
The ubiquitin molecule has several characteristics that make it particularly valuable as a carrier of peptides containing specific enzyme assaying characteristics. Native ubiquitin itself does not appear to be a suitable substrate for enzyme assays.
Therefore, while short chain peptides are commercially available for use as substrates in assaying for enzymes, they are expensive, difficult to synthesize and are not particularly useful in some assay techniques, such as being immobilized on membranes.
Assaying for enzymes, both qualitatively and quantitatively, can provide a useful diagnostic tool for examining antibodies or enzymes which, when present, may be indicative of certain diseases or other medical conditions.
The HaRAS protein, for example, has a molecular weight of about 21 KDa and is made up of 189 amino acid residues. It is an oncogene protein product which is mutant in about fifty percent of human tumors and attached to the cell surface at its C-terminus. It is known that lipid transfer enzymes, such as farnesyl-protein transferase (FPT), and methyl transfer enzymes, such as carboxyl methyltransferase, both modify RAS by reactions at the C-terminal end of the RAS molecule as will be shown below. Therefore, it is likely that a suitable clinical assay for the presence of enzymes that modify the C-terminus of RAS, either in normal or abnormal levels, can be a diagnostic tool in the science of oncology.
Protein kinases are also known to be mutated in human tumors and are active by adding phosphates on protein residues on the surface of cells.
It would therefore be highly beneficial in the art of enzymology to provide a substrate containing any multiplicity of peptide sequences which can readily and economically be cloned and utilized in a variety of assay techniques and/or diagnostic tests for enzymes which will react with such substrate.